Cytokines are essential cell signaling molecules that influence a diverse range of biological processes. Many are used as biomarkers, and the cytokine storm associated with SARS-CoV-2 infection is of particular interest in the current climate. Methods for detecting and analyzing cytokines range from single-target ELISAs through to large antibody arrays and microparticle multiplex assays, with the chosen approach being driven by specific user requirements. Here, we focus on some common challenges of cytokine research and consider effective methods for studying cytokine expression.
Cytokines perform numerous functions
Countless biological processes are accompanied by changes in cytokine expression levels. These include apoptosis, inflammation, angiogenesis, immune responses, and cellular migration, to name but a few, highlighting the critical importance of cytokines to cellular physiology. Heather Darby, field applications scientist at Luminex, reports that cytokines have also evolved into broadly accepted biomarkers of a variety of disease states and metabolic changes, making their detection and analysis a logical first step to learn more about the underlying biology.
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“Understanding how the cytokine response relates to clinical presentation and prognosis can be the key to developing effective treatments,” she says. “For example, researchers studying the cytokine storm detected in some critical patients with COVID-19 are working to identify a representative panel of relevant cytokines and track their changes in response to different states of viral infection. Multiplexing the detection of this panel of markers in a single test dramatically accelerates the speed of research in this area and may provide novel therapeutic targets or strategies.”
Detecting and analyzing cytokines can be challenging
According to Valerie Jones, Ph.D., director of marketing and sales at RayBiotech, serum and plasma are among the most commonly used sample types for cytokine research due to their close contact with most tissues and organs. “This makes their composition reflective of the overall health and functioning of the body,” she says, “although other, more tissue-specific fluids (e.g. synovial or cerebrospinal fluid) may contain valuable biomarkers pertaining to their local area.” Cell culture supernatants and tissue lysates are also frequently studied. However, Jones notes that the latter can be difficult to work with due to high levels of lipid, collagen, or other structural proteins that may produce high background signals in cytokine assays. As well as masking the detection of low-abundance targets, unwanted background signal can hinder validation efforts such as parallelism where binding factor balance is shifted by dilution.
Another inherent challenge of detecting and analyzing cytokines concerns the biological relevance of results. “Even when well-characterized reagents are available for cytokine assays, these tend to be synthetic or recombinant,” explains Darby. “Moreover, due to a lack of common and well-qualified standard material for use during development, most test and analyte-specific reagent (ASR) manufacturers end up not correlating to one another’s final concentrations.” Further complications arise from the fact that some cytokines are incredibly sensitive to sample handling and processing methods, while others (e.g. IL-12) exist in multiple isoforms that require antibody specificity be rigorously validated. It can also be difficult to find a universal dilution factor for both high- and low-abundance cytokines when multiplexing.
“Profiling cytokine release in response to localized infection can especially be problematic,” comments Marissa Beck, product manager for ELISA at Bio-Techne. “Not only can cytokines mediate locally or throughout the body, but they are typically pleiotropic and redundant—meaning that an individual cytokine can have many functions and different cytokines can share the same function.” While echoing this point, Lauren Jachimowicz, applications development scientist at Agilent Technologies, stresses that a major advantage of cytokine analysis is that it provides a measure of cells’ functional capacity. “Although the expression of specific cell surface receptors and transcription factors can be used to determine if a cell is activated, this is an indirect functional measurement,” she says. “By using techniques to determine which specific cells are producing the cytokine of interest and to what extent, researchers can gain deeper insights into a heterogeneous population.”
Which assay should you choose?
Factors to consider when selecting a suitable assay format to detect and analyze cytokines include the type of sample material, the number of analytes, and the sensitivity and throughput required. ELISA is often used for cytokine detection as it is quantitative, easy to use, and relatively inexpensive; high sensitivity ELISAs such as Quantikine® assays are also widely available. Drawbacks of ELISA are that it can have high sample volume requirements and is typically designed to measure only one cytokine at a time. “For these reasons, many researchers are moving toward automated ELISAs that eliminate user error and may be more efficient in terms of sample and reagent consumption,” observes Beck. “For example, the Simple Plex™ Ella microfluidic platform has recently been used to measure IL1β, IL6, IL8 and TNFα in parallel during a study to establish whether cytokine expression is predictive of mortality in COVID19 patients.” Microparticle multiplex assays such as Luminex and antibody arrays like those developed by RayBiotech are also extremely popular.
Image: Cytokine immunoassays are subject to potential interferences from components in complex samples like serum or plasma. Removing false positives and false negatives, such as soluble receptors, binding partners, and heterophilic antibodies, is key to obtaining the correct specificity. Image provided by Bio-Techne.
“Antibody arrays have been widely cited for cytokine research,” says Jones. “For instance, a recent study using a quantitative mouse array with 120 analytes found that Dkk1, secreted by bone marrow osteoprogenitors, promotes hematopoietic regeneration in mice. It has also been possible using a human cytokine array with 80 analytes to identify secreted molecules from CD10+ GPR77+ cancer-associated fibroblasts that contribute to chemoresistance in co-cultured tumor cells. A main advantage of antibody arrays and cytometric bead assays is that they are much more economical in terms of cost and sample use compared to ELISA. Although sensitivity may be reduced for some analytes, this is generally counterbalanced by the amount of data produced from a single experiment.”
Other techniques used for cytokine research are ELISpot, which has excellent sensitivity but is specific to measuring cytokines secreted by small populations of suspension cells, and intracellular flow cytometry detection. “Although flow cytometry-based cytokine analysis requires whole cells rather than just supernatant, it can be used to measure multiple cytokines simultaneously and allows for distinguishing and characterization of the cells producing those cytokines,” notes Jachimowicz. “This is useful to support many different areas of research, with the study of how cytokines stimulate immune cells such as T-cells, macrophages, and NK cells to kill virally infected cells being just one example.”